Inflammatory cell inhibitors

ABSTRACT

Compounds of general formula (I)                    
     wherein R 4  is an ester or thioester group and R, R 1 , R 2 , and R 3  are as specified in the description, inhibit monocyte and/or macrophage and/or lymphocyte activation and lymphocyte proliferation.

This application is a 371 of PTC/GB99/00663, Mar. 5, 1999.

The present invention relates to the use of certain esters andthioesters for the treatment of diseases responsive to inhibition ofmonocyte and/or macrophage and/or lymphocyte activation and oflymphocyte proliferation.

BACKGROUND TO THE INVENTION

Inflammatory diseases represent a large and increasing health burdenthroughout the world. Chronic inflammatory conditions include autoimmunedisorders (rheumatoid arthritis, multiple sclerosis, psoriasis),allergies, periodontists and gastrointestinal inflammatory diseases.These diseases are characterised by an influx of inflammatory cells intothe extravascular connective tissue of target organs. In these sites,aberrant activation of circulating and/or resident lymphocytes becomesself-perpetuating and this leads to chronic tissue destruction. The maincells responsible for this destruction are lymphocytes andmonocyte/macrophages. This type of inflammation may also be generated bypersistent infection (e.g., tuberculosis), in chronic rejection of solidorgan transplants and in chronic graft-versus-host disease followingbone marrow transplantation.

The recruitment and accumulation of these cells into the target site isregulated by the release of soluble chemokines and by specific adhesionmolecules expressed on the extravascular tissues and on the migratinglymphoid/myeloid cells. Activation of macrophages and the proliferationof lymphocytes, particularly T lymphocytes, within these sites leads tothe production of pro-inflammatory molecules; chemokines, cytokines,enzymes, reactive oxygen species (ROS), leukotrienes and prostaglandins.

Chemokines such as RANTES, MIP-1alpha/beta, MIP-3alpha/beta, MCP-1 toMCP-4, TARC, PARC, lymphotactin and fractalkine are released atinflammatory sites and recruit monocytes and T cells. Cytokines such asTNF, INFgamma, IL-1beta, IL-2, IL-12 and IL-18 are released which drivecell proliferation. Multiple enzymes are activated in these inflammatorycells and these include LTA4 hydrolase, 5-LO, COX-2 and PLA-2.Tissue-degrading enzymes such as metalloproteases and cysteine proteasesare also released. Gene expression of many of these molecules isregulated by the ubiquitous transcription factor NFkB. Theanti-inflammatory activity of steroids is largely through inhibition ofactivated NFkB but they also affect other pathways which results intoxic side effects. There are various modes of treatment for chronicinflammatory conditions but they largely consist of using anon-steroidal anti-inflammatory agent initially followed up by steroids,cyclosporin/FK506 or, in severe conditions, nucleoside synthesisinhibitors and alkylating agents.

In addition to chronic inflammatory diseases, there are severalclinically-important conditions associated with acute inflammation.These include acute respiratory distress syndrome (ARDS), pancreatitisand the allergic conditions of rhinitis and urticaria. Acute transplantrejection and graft-versus-host disease are also a result of rapidinflammatory responses. Lymphocytes are important in priming many acuteinflammatory responses due to antibody production (IgE, complementfixing IgG or IgM) and cytokine production but granulocytes and mastcells tend to play a more direct role in the pathogenesis. Monocyteproducts also drive acute inflammation.

Agents which can inhibit monocyte/macrophage and lymphocyte activationand subsequent lymphocyte proliferation would be useful in treatinginflammatory disorders. Such agents would reduce the number of cells inthe inflammatory site and the levels of pro-inflammatory mediators.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the finding that certain esters andthioesters have these properties, and are therefore of use for thetreatment of chronic and acute inflammatory conditions responsive tosuch inhibiton. Chronic and acute inflammatory conditions includeautoimmune disorders (eg rheumatoid arthritis, multiple sclerosis,psoriasis), allergies, periodontists, gastrointestinal inflammatorydiseases, acute respiratory distress syndrome (ARDS), pancreatitis, theallergic conditions of rhinitis and urticaria, transplant rejection andgraft-versus-host disease.

In our earlier international patent application PCT/GB97/02398 (WO98/11063), there is disclosed the use of the same class of esters andthioesters as inhibitors of the proliferation of rapidly dividing cells,and thus as agents for the treatment, inter alia, of cancer. However,the present utility as inhibitors of monocyte and/or macrophage and/orlymphocyte activation and of lymphocyte proliferation is unrelated toand not predictable from the teaching of that application.

A few patent publications (WO 92/09563, U.S. Pat. 5,183,900, U.S. Pat.5,270,326, EP-A-0489577, EP-A-0489579, WO 93/09097, WO 93/24449, WO94/25434, WO 94/25435, WO 95/04033, WO 95/19965, and WO 95/22966)include within their generic disclosure carboxylate ester compoundshaving matrix metalloproteinase inhibitory activity. In accordance withthe present invention, such compounds are now recognised to haveactivity as inhibitors of monocyte and/or macrophage and/or lymphocyteactivation and of lymphocyte proliferation, but that activity is notsuggested by, or predictable from, those publications.

WO 95/04033 disclosesN⁴-hydroxy-N′-(1-(S)-methoxycarbonyl-2,2-dimethylpropyl)-2-(R)-(4-chlorophenylpropyl)succinamideas an intermediate for the preparation of the corresponding methylamideMMP inhibitor. In addition, Int. J. Pept. Protein Res. (1996), 48(2),148-155 discloses the compound

Ph-CH₂CH(CO-Ile-OtBu)CH₂CONHOH

as an intermediate in the preparation of compounds which are inhibitorsof neurotensin-degrading enzymes. However, those two appear to be theonly specific known carboxylate ester compounds of the kind with whichthis invention is concerned.

DETAILED DESCRIPTION OF THE INVENTION

In its broadest aspect, the present invention provides a method fortreatment of mammals suffering diseases responsive to inhibition ofmonocyte and/or macrophage and/or lymphocyte activation and oflymphocyte proliferation, comprising administering to the mammalsuffering such disease an amount of a compound of general formula (I) ora pharmaceutically acceptable salt hydrate or solvate thereof sufficientto inhibit such activity:

wherein

R is hydrogen or (C₁-C₆)alkyl;

R₁ is hydrogen;

(C₁-C₆)alkyl;

(C₂-C₆)alkenyl;

phenyl or substituted phenyl;

phenyl (C₁-C₆)alkyl or substituted phenyl(C₁-C₆)alkyl;

phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl

heterocyclyl or substituted heterocyclyl;

heterocyclyl(C₁-C₆)alkyl or substituted heterocyclyl(C₁-C₆)alkyl;

a group BSO_(n)A- wherein n is 0, 1 or 2 and B is hydrogen or a (C₁-C₆)alkyl,

phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl,(C₁-C₆)acyl, phenacyl or substituted phenacyl group, and A represents(C₁-C₆)alkylene;

hydroxy or (C₁-C₆)alkoxy;

amino, protected amino, acylamino, (C₁-C₆)alkylamino ordi-(C₁-C₆)alkylamino;

mercapto or (C₁-C₆)alkylthio;

amino(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C6)alkyl,di(C₁-C₆)alkylamino(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,mercapto(C₁-C₆)alkyl or carboxy(C₁-C₆) alkyl wherein the amino-,hydroxy-, mercapto- or carboxyl-group are optionally protected or thecarboxyl-group amidated;

lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl,di(lower alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-loweralkanoylamino; or

a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring containingup to 3 heteroatoms, any of which may be (i) substituted by one or moresubstituents selected from C₁-C₆ alkyl, C₂-C₆ alkenyl, halo, cyano(—CN), —CO₂H, —CO₂R, —CONH₂, —CONHR, —CON(R)₂, —OH, —OR, oxo-, —SH, —SR,—NHCOR, and —NHCO₂R wherein R is C₁-C₆ alkyl or benzyl and/or (ii) fusedto a cycloalkyl or heterocyclic ring;

R₂ is a C₁-C₁₂ alkyl,

C₂-C₁₂ alkenyl,

C₂-C₁₂ alkynyl,

phenyl(C₁-C₆ alkyl)-,

heteroaryl(C₁-C₆ alkyl)-,

phenyl(C₂-C₆ alkenyl)-,

heteroaryl(C₂-C₆ alkenyl)-,

phenyl(C₂-C₆ alkynyl)-,

heteroaryl(C₂-C₆ alkynyl)-,

cycloalkyl(C₁-C₆ alkyl)-,

cycloalkyl(C₂-C₆ alkenyl)-,

cycloalkyl(C₂-C₆ alkynyl)-,

cycloalkenyl(C₁-C₆ alkyl)-,

cycloalkenyl(C₂-C₆ alkenyl)-,

cycloalkenyl(C₂-C₆ alkynyl)-,

phenyl(C₁-C₆ alkyl)O(C₁-C₆ alkyl)-, or

heteroaryl(C₁-C₆ alkyl)O(C₁-C₆ alkyl)-group,

any one of which may be optionally substituted by

C₁-C₆ alkyl,

C₁-C₆ alkoxy,

halo,

cyano (—CN),

phenyl or heteroaryl, or

phenyl or heteroaryl substituted by

C₁-C₆ alkyl,

C₁-C₆ alkoxy,

halo, or

cyano (—CN);

R₃ is the characterising group of a natural or non-natural a amino acidin which any functional groups may be protected; and

R₄ is an ester or thioester group,

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In another broad aspect of the invention, there is provided the use of acompound of formula (I) as defined in the immediately precedingparagraph, in the preparation of a pharmaceutical composition treatmentof mammals suffering diseases responsive to inhibition of monocyteand/or macrophage and/or lymphocyte activation and of lymphocyteproliferation.

In one particular aspect of the invention, the compound used is one ofgeneral formula (I) above wherein R, R₁, R₂, R₃ and R₄ are as definedabove with reference to formula (I), or a pharmaceutically acceptablesalt, hydrate or solvate thereof, PROVIDED THAT:

(i) when R and R₁ are hydrogen, R₂ is 4-chlorophenylpropyl, and R³ istert-butyl, then R₄ is not a methyl carboxylate ester group; and

(ii) when R and R₁ are hydrogen, R₂ is phenylmethyl, and R³ is1-methylprop-1-yl, then R₄ is not a tert-butyl carboxylate ester group.

In another particular aspect of the invention, the compound used is oneof general formula (I) above wherein:

R, R₁ and R₄ are as defined above with reference to formula (I)

R₂ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,

biphenyl(C₁-C₆ alkyl)-, phenylheteroaryl(C₁-C₆ alkyl)-,heteroarylphenyl(C₁-C₆ alkyl)-,

biphenyl(C₂-C₆ alkenyl)-, phenylheteroaryl(C₂-C₆ alkenyl)-,heteroarylphenyl(C₂-C₆ alkenyl)-,

phenyl(C₂-C₆ alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-,

biphenyl(C₂-C₆ alkynyl)-, phenylheteroaryl(C₂-C₆ alkynyl)-,heteroarylphenyl(C₂-C₆ alkynyl)-,

phenyl(C₁-C₆ alkyl)O(C₁-C₆ alkyl)-, or heteroaryl(C₁-C₆ alkyl)O(C₁-C₆alkyl)-,

any one of which may be optionally substituted on a ring carbon atom byC₁-C₆ alkyl, C₁-C₆ alkoxy, halo, or cyano (—CN); and

R₃ is C₁-C₆ alkyl, optionally substituted benzyl, optionally substitutedphenyl, optionally substituted heteroaryl; or

the characterising group of a natural a amino acid, in which anyfunctional group may be protected, any amino group may be acylated andany carboxyl group present may be amidated; or

a heterocyclic(C₁-C₆)alkyl group, optionally substituted in theheterocyclic ring;

and pharmaceutically acceptable salts, hydrates or solvates thereof.

As used herein the term “(C₁-C₆)alkyl” or “lower alkyl” means a straightor branched chain alkyl moiety having from 1 to 6 carbon atoms,including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.

The term “(C₂-C₆)alkenyl” means a straight or branched chain alkenylmoiety having from 2 to 6 carbon atoms having at least one double bondof either E or Z stereochemistry where applicable. This term wouldinclude, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

The term “C₂-C₆ alkynyl” refers to straight chain or branched chainhydrocarbon groups having from two to six carbon atoms and having inaddition one triple bond. This term would include for example, ethynyl,1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl,3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

The term “cycloalkyl” means a saturated alicyclic moiety having from 3-8carbon atoms and includes, for example, cyclohexyl, cyclooctyl,cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl.

The term “cycloalkenyl” means an unsaturated alicyclic moiety havingfrom 4-8 carbon atoms and includes, for example, cyclohexenyl,cyclooctenyl, cycloheptenyl, cyclopentenyl, and cyclobutenyl. In thecase of cycloalkenyl rings of from 5-8 carbon atoms, the ring maycontain more than one double bond.

The term “aryl” means an unsaturated aromatic carbocyclic group which ismoncyclic (eg phenyl) or polycyclic (eg naphthyl).

The unqualified term “heterocyclyl” or “heterocyclic” means (i) a 5-7membered heterocyclic ring containing one or more heteroatoms selectedfrom S, N and O, and optionally fused to a benzene ring, including forexample, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl,thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl,pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazolyl,maleimido, succinimido, phthalimido,1,2-dimethyl-3,5-dioxo-1,2,4-triazolidin4-yl,3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl,2-methyl-3,5-dioxo-1,2,4-oxadiazol4-yl,3-methyl-2,4,5-trioxo-1-imidazolidinyl,2,5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl,2,5-dioxo-1-pyrrolidinyl or 2,6-dioxopiperidinyl, or (ii) anaphththalimido (ie 1,3-dihydro-1,3-dioxo-2H-benz[f]isoindol-2-yl),1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl,1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or2,3-dihydro-1,3-dioxo-1H-benz[d,e]isoquinolin-2-yl group.

The term “heteroaryl” means a 5-7 membered substituted or unsubstitutedaromatic heterocycle containing one or more heteroatoms. Illustrative ofsuch rings are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, trizolyl, thiadiazolyl,oxadiazolyl, pyridinyi, pyridazinyl, pyrimidinyl, pyrazinyl andtriazinyl.

The term “ester” or “esterified carboxyl group” means a group R₉O(C═O)—in which R₉ is the group characterising the ester, notionally derivedfrom the alcohol R₉OH.

The term “thioester” means a group R₉S(C═O)— or R₉S(C═S)— or R₉O(C═S)—in which R₉ is the group characterising the thioester, notionallyderived from the alcohol R₉OH or the thioalcohol R₉SH.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four substituents, each of which independently may be (C₁-C₆)alkyl,(C₁-C₆)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo(including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl,—COOH, —CONH₂, —CN, —COOR^(A), —CONHR^(A) or —CONHR^(A)R^(A) whereinR^(A) is a (C₁-C₆)alkyl group or the residue of a natural alpha-aminoacid.

The term “side chain of a natural or non-natural alpha-amino acid” meansthe group R¹ in a natural or non-natural amino acid of formulaNH₂—CH(R¹)—COOH.

Examples of side chains of natural alpha amino acids include those ofalanine, arginine, asparagine, aspartic acid, cysteine, cystine,glutamic acid, histidine, 5-hydroxylysine, 4-hydroxyproline, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyricacid, 3,4-dihydroxyphenylalanine, homoserine, α-methylserine, ornithine,pipecolic acid, and thyroxine.

Natural alpha-amino acids which contain functional substituents, forexample amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, orindolyl groups in their characteristic side chains include arginine,lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine,threonine, tyrosine, and cysteine. When R₃ in the compounds of theinvention is one of those side chains, the functional substituent mayoptionally be protected.

The term “protected” when used in relation to a functional substituentin a side chain of a natural alpha-amino acid means a derivative of sucha substituent which is substantially non-functional. For example,carboxyl groups may be esterified (for example as a C₁-C₆ alkyl ester),amino groups may be converted to amides (for example as a NHCOC₁-C₆alkyl amide) or carbamates (for example as an NHC(═O)OC₁-C₆ alkyl orNHC(═O)OCH₂Ph carbamate), hydroxyl groups may be converted to ethers(for example an OC₁-C₆ alkyl or a O(C₁-C₆ alkyl)phenyl ether) or esters(for example a OC(═O)C₁-C₆ alkyl ester) and thiol groups may beconverted to thioethers (for example a tert-butyl or benzyl thioether)or thioesters (for example a SC(═O)C₁-C₆ alkyl thioester).

Examples of side chains of non-natural alpha amino acids include thosereferred to below in the discussion of suitable R₃ groups for use incompounds of the present invention.

Salts of the compounds used in the invention include physiologicallyacceptable acid addition salts for example hydrochiorides,hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates,phosphates, acetates, citrates, succinates, lactates, tartrates,fumarates and maleates. Salts may also be formed with bases, for examplesodium, potassium, magnesium, and calcium salts.

There are several chiral centres in the compounds used according to theinvention because of the presence of asymmetric carbon atoms. Thepresence of several asymmetric carbon atoms gives rise to a number ofdiastereomers with R or S stereochemistry at each chiral centre. Forexample, in the compounds used in the invention, the C atom carrying thehydroxamic acid and R₁ groups may be in the R or S configuration, the Catom carrying the R₂ group may be predominantly in the R configuration,and the C atom carrying the R₃ and R₄ groups may be in either the R or Sconfiguration, with the predominantly S configuration presentlypreferred.

As mentioned above, compounds of formula (I) above, are useful in humanor veterinary medicine since they inhibit monocyte/macrophage andlymphocyte activation and subsequent lymphocyte proliferation. They aretherefore useful for the treatment of chronic and acute inflammatoryconditions, including autoimmune disorders (eg rheumatoid arthritis,multiple sclerosis, psoriasis), allergies, periodontists,gastrointestinal inflammatory diseases, acute respiratory distresssyndrome (ARDS), pancreatitis, the allergic conditions of rhinitis andurticaria, acute transplant rejection and graft-versus-host disease.

The compounds with which the invention is concerned may be prepared foradministration by any route consistent with their pharmacokineticproperties.

Orally administrable compositions may be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions.Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art,for example as described in standard textbooks of pharmaceutics such asthe British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterilemedium. Depending on the vehicle and concentration used, the drug caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as a local anaesthetic, preservative and buffering agentscan be dissolved in the vehicle.

Clinically safe and effective dosages for the compounds with which theinvention is concerned will be determined by clinical trials, as isrequired by the regulatory authorities in the art. It will be understoodthat the specific dose level for any particular patient will depend upona variety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

In the compounds used in the invention, examples of substituents R, toR₄ are given below:

The group R₁

R₁ may be, for example,

hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, hydroxyl, methoxy,allyl, phenylpropyl, phenylprop-2-enyl, thienylsulphanylmethyl,thienylsulphinylmethyl, or thienylsulphonylmethyl; or

C₁-C₄ alkyl,eg methyl, ethyl n-propyl or n-butyl, substituted by aphthalimido, 1,2-dimethyl-3,5-dioxo-1,2,4-triazolidin4-yl,3-methyl-2,5-dioxo-1-imidazolidinyl,3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl,2-methyl-3,5-dioxo-1,2,4-oxad iazol4-yl,3-methyl-2,4,5-trioxo-1-imidazolidinyl,2,5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl,2,5-dioxo-1-pyrrolidinyl or 2,6-dioxopiperidinyl,5,5-dimethyl-2,4-dioxo-3-oxazolidinyl,hexahydro-1,3-dioxopyrazolo[1,2,a][1,2,4]-triazol-2-yl, or anaphththalimido (ie 1,3-dihydro-1,3-dioxo-2H-benz[f]isoindol-2-yl),1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl,1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or2,3-dihydro-1,3-dioxo-1H-benz[d,e]isoquinolin-2-yl group; or

cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl,cyclopropyl, tetrahydropyranyl or morpholinyl.

Presently preferred R₁ groups include n-propyl, allyl, hydroxy, methoxyand thienylsulfanylmethyl.

The group R₂

R₂ may for example be

C₁-C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)-optionally substituted in the phenyl ring;

heteroaryl(C₁-C₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆alkynyl)- optionally substituted in the heteroaryl ring;

4-phenylphenyl(C₁-C₆ alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-,4-phenylphenyl(C₃-C₆ alkynyl)-, 4-heteroarylphenyl(C₁-C₆ alkyl)-,4-heteroarylphenyl(C₃-C₆ alkenyl)-, 4-heteroarylphenyl(C₃-C₆ alkynyl)-,optionally substituted in the terminal phenyl or heteroaryl ring;

phenoxy(C₁-C₆ alkyl)- or heteroaryloxy(C₁-C₆ alkyl)- optionallysubstituted in the phenyl or heteroaryl ring;

Specific examples of such groups include methyl, ethyl, n- andiso-propyl, n-, iso- and tert-butyl, n-pentyl, n-hexyl, n-heptyl,n-nonyl, n-decyl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl,3-(2-chlorophenyl)prop-2-yn-1-yl, phenylpropyl, 4-chlorophenylpropyl,4-methylphenylpropyl, 4-methoxyphenylpropyl, phenoxybutyl,3-(4-pyridylphenyl)propyl-, 3-(4-(4-pyridyl)phenyl)prop-2-yn-1-yl,3-(4-phenylphenyl)propyl-, 3-(4-phenyl)phenyl)prop-2-yn-1-yl and3-[(4-chlorophenyl)phenyl]propyl-, cyclopentylmethyl, and benzyl.

Presently preferred R₂ groups include n- and iso-butyl, n-hexyl,cyclopentylmethyl, benzyl, and 3-(2-chlorophenyl)prop-2-yn-1-yl.

The group R₃

R₃ may for example be C₁-C₆ alkyl, phenyl, 2, -3-, or 4-hydroxyphenyl,2, -3-, or 4-methoxyphenyl, 2- or 3-thienyl, 2, -3-, or 4-pyridylmethyl,benzyl, 2, -3-, or 4-hydroxybenzyl, 2, -3-, or 4-benzyloxybenzyl, 2,-3-, or 4-C₁-C₆ alkoxybenzyl, or benzyloxy(C₁-C₆alkyl)-group; or

the characterising group of a natural α amino acid, in which anyfunctional group may be protected, any amino group may be acylated andany carboxyl group present may be amidated; or

a group -[Alk]_(n)R₆ where Alk is a (C₁-C₆)alkyl or (C₂-C₆)alkenyl groupoptionally interrupted by one or more —O—, or —S— atoms or —N(R₇)—groups [where R₇ is a hydrogen atom or a (C₁-C₆)alkyl group], n is 0 or1, and R₆ is an optionally substituted cycloalkyl or cycloalkenyl group;or

a benzyl group substituted in the phenyl ring by a group of formula—OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid halide,ester or amide derivative thereof, said residue being linked via anamide bond, said amino acid being selected from glycine,

α or β alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, serine, threonine, cysteine, methionine, asparagine,glutamine, lysine, histidine, arginine, glutamic acid, and asparticacid; or

a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- ordi-substituted in the heterocyclic ring with halo, nitro, carboxy,(C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl,hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino,mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or(C₁-C₆)alkylphenylmethyl; or

a group —CR_(a)R_(b)R_(c) in which:

each of R_(a), R_(b) and R_(c) is independently hydrogen, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl;or

R_(c) is hydrogen and R_(a) and R_(b) are independently phenyl orheteroaryl such as pyridyl; or

R_(c) is hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,phenyl(C₁-C₆)alkyl, or (C₃-C₈)cycloalkyl, and R_(a) and R_(b) togetherwith the carbon atom to which they are attached form a 3 to 8 memberedcycloalkyl or a 5- to 6-membered heterocyclic ring; or

R_(a), R_(b) and R_(c) together with the carbon atom to which they areattached form a tricyclic ring (for example adamantyl); or

R_(a) and R_(b) are each independently (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group as defined for R_(c)below other than hydrogen, or R_(a) and R_(b) together with the carbonatom to which they are attached form a cycloalkyl or heterocyclic ring,and R_(c) is hydrogen, —OH, —SH, halogen, —CN, —CO₂H,(C₁-C₄)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl,—S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl, —SO₂(C₂-C₆)alkenyl or a group —Q-Wwherein Q represents a bond or —O—, —S—, —SO—or —SO₂— and W represents aphenyl, phenylalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl,(C₄-C₈)cycloalkenyl, (C₄-C₈)cycloalkenylalkyl, heteroaryl orheteroarylalkyl group, which group W may optionally be substituted byone or more substituents independently selected from, hydroxyl, halogen,—CN, —CO₂H, —CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl,—CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl,—S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂,—NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl,phenyl or benzyl.

Examples of particular R₃ groups include benzyl, phenyl,cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl, iso-butyl,sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl,1-methylthio-1-methylethyl, and 1-mercapto-1-methylethyl.

Presently preferred R₃ groups include phenyl, benzyl, tert-butoxymethyland iso-butyl.

The group R₄

Examples of particular ester and thioester groups R₄ groups includethose of formula —(C═O)OR₉, —(C═O)SR₉, —(C═S)SR₉, and —(C═S)OR₉, whereinR₉ is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, cycloalkyl, cycloalkyl(C₁-C₆)alkyl-,phenyl, heterocyclyl, phenyl(C₁-C₆)alkyl-, heterocyclyl(C₁-C₆)alkyl-,(C₁-C₆)alkoxy(C₁-C₆)alkyl-, (C₁-C₆)alkoxy(C₁-C₆)alkoxy(C₁-C₆)alkyl-, anyof which may be substituted on a ring or non-ring carbon atom or on aring heteroatom, if present. Examples of such R₉ groups include methyl,ethyl, n-and iso-propyl, n-, sec- and tert-butyl, 1-ethyl-prop-1-yl,1-methyl-prop-1-yl, 1-methyl-but-1-yl, cyclopentyl, cyclohexyl, allyl,phenyl, benzyl, 2-, 3- and 4-pyridylmethyl, N-methylpiperidin-4-yl,1-methylcyclopent-1-yl, adamantyl, tetrahydrofuran-3-yl andmethoxyethyl.

Presently preferred are compounds of formula (I) wherein R₄ is acarboxylate ester of formula —(C═O)OR₉, wherein R₉ is benzyl,cyclopentyl, isopropyl or tert-butyl.

The group R

Presently preferred R groups are hydrogen and methyl.

Specific compounds for use in accordance with the invention includethose of examples 1, 2, 3, 6, 19, 39, 40 and 43.

Compounds used according to the present invention may be prepared by themethods described in our published international patent application NoWO 98/11063. Specific examples of compounds which may be used are thoseof the following examples 1-50. Examples 1-42 are compounds disclosed inWO 98/11063.

EXAMPLE 1 (Example 1 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid methyl ester.

EXAMPLE 2 (Example 2 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid ethyl ester.

EXAMPLE 3 (Example 3 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid isopropyl ester.

EXAMPLE 4 (Example 5 of WO 98/11063)

3R-(2-Phenyl-1S-methylcarboxy-ethylcarbamoyl)-2S,5-dimethylhexanohydroxamic acid

EXAMPLE 5 (Example 6 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenyl-propionicacid tert-butyl ester

EXAMPLE 6 (Example 7 of WO 98/11063)

2S-(2R-Hydroxycarbamoylmethyl-4-methyl-pentanoylamino)-3-phenyl-propionicacid isopropyl ester

EXAMPLE 7 (Example 8 of WO 98/11063)

2S-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamine]-3-phenyl-propionicacid isopropyl ester.

EXAMPLE 8 (Example 9 of WO 98/11063)

2S-[2R-(1S-Hydroxycarbamoyl-ethyl)-4-methyl-pentanoylamino]-3-phenyl-propionicacid isopropyl ester.

EXAMPLE 9 (Example 10 of WO 98/11063)

2S-(2R-Hydroxycarbamoylmethyl-octanoylamino)-3-phenyl-propionic acidisopropyl ester.

EXAMPLE 10 (Example 11 of WO 98/11063)

2S-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamino]-3-phenyl-propionicacid cyclopentyl ester.

EXAMPLE 11 (Example 12 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3S-methyl-pentanoicacid cyclopentyl ester.

EXAMPLE 12 (Example 13 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid 2-methoxy-ethyl ester.

EXAMPLE 13 (Example 1 of WO 98/11063)

2S-[2R-(1S-Hydroxycarbamoyl-ethyl)-4-methyl-pentanoylamino]-3-phenyl-propionicacid 2-methoxy-ethyl ester.

EXAMPLE 14 (Example 15 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hexanoylamino)-3,3-dimethyl-butyricacid 2-methoxy-ethyl ester.

EXAMPLE 15 (Example 1 of WO 98/11063)

2S-[2R-(S-Hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3-phenyl-propionicacid isopropyl ester.

EXAMPLE 16 (Example 17 of WO 98/11063)

2S-{2R-[1S-Hydroxycarbamoyl-2-(thiophen-2-ylsulphanyl)-ethyl]4-methyl-pentanoylamino}-3-phenyl-propionicacid isopropyl ester.

EXAMPLE 17 (Example 18 of WO 98/11063)

2S-[2-R-(1S-Hydroxycarbamoyl-ethyl)-4-methyl-pentanoylamino]-3,3-dimethyl-butyricacid 2-methoxy-ethyl ester.

EXAMPLE 18 (Example 19 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3,3-dimethyl-butyricacid 2-methoxy-ethyl ester.

EXAMPLE 19 (Example 20 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid cyclopentyl ester.

EXAMPLE 20 (Example 21 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hexanoylamino)-3-phenylpropionicacid isopropyl ester.

EXAMPLE 21 (Example 22 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3,3-dimethyl-butyricacid isopropyl ester.

EXAMPLE 22 (Example 23 of WO 98/11063)

2R-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid isopropyl ester.

EXAMPLE 23 (Example 24 of WO 98/11063)

2S-[2R-(S-Hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3-dimethyl-butyricacid isopropyl ester.

EXAMPLE 24 (Example 25 of WO 98/11063)

2S-{(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoyl)-methyl-amino)-3-phenylpropionicacid isopropyl ester.

EXAMPLE 25 (Example 26 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid benzyl ester.

EXAMPLE 26 (Example 27 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-4-methyl-pentanoicacid cyclopentyl ester.

EXAMPLE 27 (Example 28 of WO 98/11063)

3-Cyclohexyl-2S-(3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-propionicacid cyclopentyl ester.

EXAMPLE 28 (Example 29 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid 1-methyl-piperidin-4-yl ester.

EXAMPLE 29 (Example 30 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid 1-ethyl-propyl ester.

EXAMPLE 30 (Example 31 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid 1S-methyl-butyl ester.

EXAMPLE 31 (Example 32 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid cyclohexyl ester.

EXAMPLE 32 (Example 1of WO 98/11063)

2S{2R-[1S-Hydroxycarbamoyl-2-(thiophen-2-ylsulphanyl)-ethyl]-4-methyl-pentanoylamino}-3,3-dimethyl-butyricacid isopropyl ester.

EXAMPLE 33 (Example 34 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid 1R-methyl-butyl ester.

EXAMPLE 34 (Example 35 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-phenylpropionicacid tetrahydro-furan-3(R, S)-yl ester.

EXAMPLE 35 (Example 36 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3,3-dimethyl-butyricacid cyclopentyl ester.

EXAMPLE 36 (Example 37 of WO 98/11063)

2S-[2R-(1S-Cyclopentyl-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamino]-3-phenyl-propionicacid cyclopentyl ester.

EXAMPLE 37 (Example 38 of WO 98/11063)

2S-[2R-(1S-Hydroxy-hydroxycarbamoyl-methyl)-pent4-ynoylamino]-3-phenylpropionicacid cyclopentyl ester.

EXAMPLE 38 (Example 39 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-3-pyridin-3-yl-propionicacid cyclopentyl ester.

EXAMPLE 39 (Example 40 of WO 98/11063)

3-tert-Butoxy-2S-(3S-hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-propionicacid cyclopentyl ester.

EXAMPLE 40 (Example 41 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-phenylethanoicacid cyclopentyl ester.

EXAMPLE 41 (Example 42 of WO 98/11063)

2S-[5-(2-Chlorophenyl)-2R-(1S-hydroxy-hydroxycarbamoyl-methyl)-pent-4-ynoylamino]-3-phenylpropionicacid cyclopentyl ester.

EXAMPLE 42 (Example 43 of WO 98/11063)

2S-(3S-Hydroxycarbamoyl-2R-isobutyl-6-phenyl-hex-5-enoylamino)-3-phenyl-propionicacid cyclopentyl ester.

EXAMPLE 43

2-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-4-methyl-pentanoylamine]-2-phenyl-ethanoicacid cyclopentyl ester

Prepared using procedures similar to those described in example 8 of WO98/11063, using phenylglycine cyclopentyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 7.4-7.29 (5H, m), 5.43 (1H, s), 5.2-5.14 (1H, m), 4.02(1H, d, J=6.9 Hz), 2.94-2.85 (1H, m), 1.91-1.34 (10H, bm), 1.25-1.14(1H, m) and 0.86 (6H, dd, J=6.5, 11.5 Hz). ¹³C-NMR; δ (MeOD), 175.6,171.8, 171.4, 137.8, 129.8, 129.4, 128.6, 80.0, 73.2, 58.5, 49.2, 39.1,33.3, 33.3, 26.8, 24.5, 24.4, 23.7 and 22.1.

Diastereoisomer B

¹H-NMR; δ (MeOD), 7.33-7.19 (5H, m), 5.3 (1H, s), 5.11-5.06 (1H, m),3.81 (1H, d, J=7.3 Hz), 2.83-2.74 (1H, m), 1.83-1.45 (10H, bm),1.12-1.03 (1H, m) and 0.88-0.81 (6H, dd, J=6.4, 12.3 Hz). ¹³C-NMR; δ(MeOD), 175.8, 171.8, 171.5, 137.3, 129.8, 129.5, 128.8, 79.9, 73.3,58.7, 48.9, 39.2, 33.3, 33.3, 26.7, 24.5, 24.5, 24.0 and 22.2.

EXAMPLE 44

2-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-phenylethanoicacid isopropyl ester

Prepared using methods similar to those described in example 41 ofapplication WO 98/11063 using phenylglycine isopropyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 7.34-7.24 (5H, m), 5.59-5.42 (1H, m), 5.36 (1H, s),5.02-4.77 (3H, m), 2.63-2.53 (1H, m), 2.17-2.02 (2H, m), 1.89-1.78 (1H,m), 1.63-1.45 (2H, m), 1.18 (3H, d, J=6.3 Hz), 1.05 (3H, d, J=6.2 Hz),1.00-0.93 (1H, m), 0.88 (3H, d, J=6.5 Hz) and 0.81 (3H, d, J=6.5 Hz).¹³C-NMR; δ (MeOD), 176.2, 172.4, 171.3, 137.6, 136.0, 129.9,129.6,129.0,117.4, 70.5, 58.7,47.4, 41.5, 36.0, 26.7, 24.5, 21.9, 21.7 and21.7.

Diastereoisomer B

¹H-NMR; δ (MeOD), 7.4-7.34 (5H, m), 5.77-5.61 (1H, m), 5.42 (1H, s),5.1-4.98 (3H, m), 2.7-2.6 (1H, m), 2.44-2.17 (3H, m), 1.61-1.5 (1H, m),1.42-1.29 (1H, m), 1.25 (3H, d, J=6.3 Hz), 1.13 (3H, d, J=6.2 Hz),1.09-1.00 (1H, m) and 0.81 (6H, d, J=6.4 Hz). ¹³C-NMR; δ (MeOD), 176.4,172.5, 171.5, 137.2, 136.4, 129.9, 129.6, 129.0, 117.5, 70.5, 58.8,48.4, 47.4, 41.3, 36.0, 27.1, 24.3, 21.9, 21.8 and 21.6.

EXAMPLE 45

2-[2R-(S-Hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3-phenylethanoicacid cyclopentyl ester

Prepared using methods similar to those described in example 16 ofapplication WO 98/11063, using phenylglycine cyclopentyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 8.83 (1H, d, J=6.6 Hz), 7.48-7.29 (5H, m), 5.44-5.42(1H, m), 5.20-5.16 (1H, m), 3.53 (1H, d, J=9.7 Hz), 3.17 (3H, s),2.89-2.79 (1H, m), 1.90-1.54 (10H, bm), 1.06-0.99 (1 H, m), 0.95 (3H, d,J=6.5 Hz) and 0.90 (3H, d, J=6.4 Hz). ¹³C-NMR; δ (MeOD), 175.3, 171.6,169.4, 137.5, 129.7, 129.4, 128.7, 83.1, 79.9, 58.7, 58.1, 48.5, 38.4,33.4, 33.3, 26.7, 24.6, 24.5, 24.3 and 21.8.

Diastereoisomer B

¹H-NMR; δ (MeOD), 7.39-7.30 (5H, m), 5.45 (1H, s), 5.21-5.15 (1H, m),3.59 (1H, d, J=9.4 Hz), 3.29 (3H, s), 2.89-2.79 (1H, m), 1.93-1.49 (9H,bm), 1.42-1.21 (1H, m), 1.01 (1H, ddd, J=3.7, 9.9, 13.3 Hz), 0.83 (3H,d, J=6.5 Hz) and 0.79 (3H, d, J=6.6 Hz). ¹³C-NMR; δ (MeOD), 175.1,171.5, 169.5, 137.9, 129.7, 129.4, 128.7, 83.0, 79.8, 58.5, 58.3, 48.6,38.5, 33.3, 27.8, 24.5, 24.4, 24.1 and 21.7.

EXAMPLE 46

2-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(4-methoxyphenyl)ethanoicacid cyclopentyl ester

Prepared using methods similar to those described in example 41 ofapplication WO 98/11063, using 4-methoxyphenylglycine cyclopentyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 8.94 (1H, d, J=6.4 Hz), 7.32 (2H, d, J=8.7 Hz), 6.93(2H, d, J=8.7 Hz), 5.67-5.50 (1H, m), 5.36-5.33 (1H, m), 5.20-5.14 (1H,m), 4.93-4.87 (2H, m), 3.79 (3H, s), 2.68-2.59 (1H, m), 2.24-2.09 (2H,m), 1.97-1.55 (11H, bm), 1.11-1.00 (1H, m), 0.95 (3H, d, J=6.5 Hz) and0.88 (3H, d, J=6.5 Hz). ¹³C-NMR; (MeOD), 176.2, 172.4, 171.9, 161.4,136.0, 130.2, 129.4, 117.4, 115.2, 79.7, 58.2, 55.8, 48.3, 47.3, 41.5,36.0, 33.4, 33.3, 26.7, 24.6, 24.5 and 21.7.

Diastereoisomer B

¹H-NMR; δ (MeOD), 8.96 (1H, d, J=6.7 Hz), 7.29 (2H, d, J=8.7 Hz), 6.93(2H, d, J=8.7 Hz), 5.77-5.61 (1H, m), 5.32 (1H, s), 5.20-5.15 (1H, m),5.09-4.97 (2H, m), 3.80 (3H, s), 2.64 (1H, dt, J=3.3, 11.4, 13.5 Hz),2.43-2.16 (3H, m), 1.91-1.49 (9H, bm), 1.42-1.29 (1H, m), 1.05 (1H, ddd,J=3.3, 10.1, 13.2 Hz) and 0.81 (6H, d, J=6.5 Hz). ¹³C-NMR; δ (MeOD),176.3, 172.5, 172.0, 161.4, 136.4, 130.2, 129.0, 117.5,115.2, 79.8,58.2, 55.8, 48.4, 47.4, 41.3, 36.1, 33.4, 27.1, 24.5, 24.3 and 21.6.

EXAMPLE 47

2-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(thien-2-yl)ethanoicacid cyclopentyl ester

Prepared using methods similar to those described in example 41 ofapplication WO 98/11063, using thien-2-ylglycine cyclopentyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 7.41 (1H, dd, J=5.1, 1.2 Hz), 7.12 (1H, d, J=3.5 Hz),7.01 (1H, dd, J=5.1, 3.5 Hz), 5.72 (1H, s), 5.69-5.52 (1H, m), 5.26-5.18(1H, m), 5.00-4.89 (2H, m), 2.70-2.59 (1H, m), 2.28-2.13 (2H, m),2.09-1.50 (11H, m), 1.05 (1H, ddd, J=13.8, 11.0, 2.9 Hz), 0.93 (3H, d,J=6.4 Hz) and 0.87 (3H, d, J=6.5 Hz). ¹³C-NMR; δ (MeOD), 176.5, 172.7,171.1, 139.5, 136.4,128.4, 128.3, 127.7, 117.9, 80.7, 54.1, 48.7, 47.7,41.9, 36.5, 33.8, 33.7, 27.2, 25.1, 25.0, 24.9, and 22.1.

Diastereoisomer B

¹H-NMR; δ (MeOD), 7.42 (1H, dd, J=5.0, 0.7 Hz), 7.10 (1H, d, J=3.6 Hz),7.01 (1H, dd, J=5.0, 3.6 Hz), 5.79-5.59 (2H, m), 5.28-5.19 (1H, m),5.10-4.94 (2H, m), 2.71-2.59 (1H, m), 2.36-2.16 (3H, m), 1.97-1.34 (10H,m), 1.13-1.00 (1H, m), 0.86 (3H, d, J=6.2 Hz) and 0.84 (3H, d, J=6.3Hz). ¹³C-NMR; δ (MeOD), 176.7, 172.8, 171.2, 139.3, 136.7, 128.3, 128.2,127.6, 117.9, 80.7, 54.2, 48.8, 47.8, 41.7, 36.4, 33.8, 27.5, 25.1,25.0, 24.8 and 22.1.

EXAMPLE 48

2-(3S-Hydroxycarbamoyl-2R-isobutyl-hex-5-enoylamino)-2-(thien-3-yl)ethanoicacid cyclopentyl ester

Prepared using methods similar to those described in example 41 ofapplication WO 98/11063, using thien-3-ylglycine cyclopentyl ester.

Diastereoisomer A

¹H-NMR; δ (MeOD), 7.48-7.42 (2H, m), 7.13 (1H, dd, J=4.2, 2.0 Hz),5.69-5.52 (2H, m), 5.21-5.16 (1H, m), 4.98-4.90 (2H, m), 2.71-2.59 (1H,m), 2.28-2.11 (2H, m), 2.00-1.50 (11H, m), 1.12-0.98 (1H, m), 0.94 (3H,d, J=6.4 Hz) and 0.88 (3H, d, J=6.5 Hz). ¹³C-NMR; δ (MeOD), 176.6,172.8, 171.8, 137.8, 136.4, 128.3, 128.0, 125.2, 117.9, 80.3, 54.6,41.9, 36.5, 33.8, 33.8, 27.1, 25.0, 24.9 and 22.1.

Diastereoisomer B

¹H-NMR; δ (MeOD), 7.45 (1H, dd, J=4.9, 3.0 Hz), 7.43-7.40 (1H, m), 7.12(1H, dd, J=5.0, 1.3 Hz), 5.68 (1H, ddt, J=17.0, 10.1, 6.8 Hz), 5.53 (1H,s), 5.23-5.17 (1H, m), 5.10-4.96 (2H, m), 2.70-2.60 (1H, m), 2.41-2.16(3H, m), 1.94-1.49 (9H, m), 1.44-1.29 (1H, m), 1.05 (1H, ddd, J=12.9,10.3, 3.3 Hz), 0.84 (3H, d, J=6.5 Hz) and 0.83 (3H, d, J=6.5 Hz).

EXAMPLE 49

2S-[2R-(S-Hydroxy-hydroxycarbamoyl-methyl)-3-phenyl-propanoylamine]-2-phenyl-ethanoicacid cyclopentyl ester.

This compound was prepared using the method of example 11 of patentapplication WO 98/11063 and intermediates similar to those described inpatent application WO 95/19956. ¹H-NMR; δ (MeOD), 7.39-7.15 (10H, m),5.32 (1H, s), 5,15-5.06 (1H, m), 4.05 (1H, d, J=5.7 Hz), 3.19-3.10 (1H,m), 3.02-2.81 (2H, m) and 1.89-1.40 (8H, m). ¹³C-NMR; δ (MeOD), 175.4,172.1, 171.8, 140.2, 137.9, 130.6, 130.1, 129.9, 129.7, 128.9, 80.4,72.7, 58.6, 52.4, 36.5, 33.7, 24.9 and 24.8.

EXAMPLE 50

2S-(3S-Hydroxycarbamoyl-2R-cyclopentylmethyl-hex-5-enoylamino)-2-phenyl-ethanoicacid cyclopentyl ester.

This compound was prepared using the method described for example 1 ofpatent application WO 98/11063 and intermediates similar to thosedescribed in patent application WO 94/21625. ¹H-NMR; δ (MeOD), 9.02 (1H,m), 7.40 (5H, m), 5.60 (1H, m), 5.45 (1H, m), 5,17 (1H, m), 4.90 (2H,m), 2.61 (1H, m), 2.20 (2H, m), 2.05-1.40 (17H, m) and 1.10 (3H, m).

Biological Example A

Inhibition of Mitogen-induced Lymphocyte Proliferation

The compounds of examples 1, 2, 3, 6, 19, 39, 40 and 43 above weretested for their effects on the proliferation of peripheral bloodmononuclear cells (PBMCs) in vitro. The PBMCs were activated withPokeweed mitogen (PWM), a potent activator of lymphocytes and monocyteswhich leads to B and T lymphocyte proliferation.

Human PBMCs (2×10⁴ cells/well), purified from whole blood bycentrifugation through ficoll/hypaque, were incubated with PWM (2.5μg/ml) and compounds 1 and 2 at a range of dilutions in 96 wellmicrotitre plates for 48 hrs at 37° C. in a 5% CO₂ incubator. DMSO wasrun in each assay as a vehicle control. 0.5 μCuries of tritiatedthymidine in 40 μl (Amersham) were added to each well during the final18 hrs. The cells were then harvested on glass fibre pads using aTomtech cell harvester and, after adding scintillant to the pad(Meltilex) they were placed in a Wallac MicroBeta plate counter. Theresults obtained are in counts per minute and reflect the level oftritiated thymidine incorporation into the cells and therefore the levelof cell proliferation. The percentage proliferation in test wellscompared to control wells was calculated and the inhibition curvesplotted. The IC₅₀ values were calculated from the inhibition curves. Allcompounds inhibited cell proliferation in a dose related fashion (seeTables 1 and 1 A below).

Biological Example B

Inhibition of Antigen-induced T Lymphocyte Proliferation

The compounds of examples 3, 19 and 1 above were tested for theireffects on antigen-driven proliferation of peripheral blood mononuclearcells (PBMCs) in vitro. The PBMCs were activated with Purified proteinderivative of Mycobacterium tuberculosis (PPD) which activates PPD(antigen)-specific T lymphocytes. In this system, monocytes are requiredto internalise and process the antigen and present peptides on MHC classII molecules to PPD-specific T cells for activation in addition toproviding co-stimulatory signals to the T cell (B7.1/2, CD40 ligand,etc).

Human PBMCs (2×10⁵ cells/well), purified from whole blood bycentrifugation through ficoll/hypaque, were incubated with PPD (1 μg/ml)and compounds 3, 20 or 1 at a range of dilutions in 96 well microtitreplates for 72 hrs at 37° C. in a 5% CO₂ incubator. DMSO was run in eachassay as a vehicle control. 0.5 μCuries of tritiated thymidine in 40 μl(Amersham) were added to each well during the final 18 hrs. The cellswere then harvested on glass fibre pads using a Tomtech cell harvesterand, after adding scintillant to the pad (Meltilex) they were placed ina Wallac MicroBeta plate counter. The results obtained are in counts perminute and reflect the level of tritiated thymidine incorporation intothe cells and therefore the level of cell proliferation. The percentageproliferation in test wells compared to control wells was calculated.The data was plotted and the IC₅₀ values estimated. All three compoundsinhibited cell proliferation in a dose related fashion (see Table 1below)

Biological Example C

Inhibition of Anti-CD3-induced T Lymphocyte Activation and Proliferation

The compounds of examples 1, 6, 19, 39, 40 and 43 above were tested forits effects on anti-CD3-driven proliferation of peripheral bloodmononuclear cells (PBMCs) in vitro. The PBMCs were activated withanti-CD3 antibody which directly activates T lymphocytes via the T cellreceptor CD3 component, although other cells do contribute to T cellproliferation by co-stimulation and growth factor production. HumanPBMCs (5×10⁴ cells/well), purified from whole blood by centrifugationthrough ficoll/hypaque, were incubated in monoclonal anti-CD3antibody-coated wells (5 μg/ml) and compound 1 at a range of dilutionsin a 96 well microtitre plate for 48 hrs at 37° C. in a 5% CO₂incubator. DMSO was run in each assay as a vehicle control. 0.5 μCuriesof tritiated thymidine in 40 μl (Amersham) were added to each wellduring the final 18 hrs. The cells were then harvested on glass fibrepads using a Tomtech cell harvester and, after adding scintillant to thepad (Meltilex) they were placed in a Wallac MicroBeta plate counter. Theresults obtained are in counts per minute and reflect the level oftritiated thymidine incorporation into the cells and therefore the levelof cell proliferation. The percentage proliferation in test wellscompared to control wells was calculated. The results were plotted andthe IC₅₀ value calculated. The compounds inhibited cell proliferation ina dose related fashion (see Tables 1 and 1 A below).

CD69 is a membrane marker of T cell activation. The level of CD69expression on the membranes of T cells activated by anti-CD3 wastherefore measured by fluorescence activated cell (FACS) flow cytometry.PBMCs treated with the compound of example 1 for 4 hrs showed reducedexpression of CD69 (48% of T cells) compared to untreated cells (68%).This demonstrates a significant reduction in T cell activation.

TABLE 1 PBMCs IC₅₀ (μM) stimulated EXAMPLE EXAMPLE EXAMPLE EXAMPLE with:1 2 3 19 PWM 4.7 4.8 2.4 not tested PPD 2.0 not tested 1.8 1.1 ANTI-CD32.1 not tested not tested not tested

TABLE 1A PBMCs PBMCs STIMULATED STIMULATED WITH PWM WITH ANTI-CD3COMPOUNDS IC50 (μM) IC50 (μM) EXAMPLE 19 2.1 0.9 EXAMPLE 6 5.9 6.0EXAMPLE 43 2.4 4.9 EXAMPLE 39 2.8 1.8 EXAMPLE 40 0.9 1.8

Biological Example D

Inhibition of Production of TNF

The compound of example 3 above was tested for its effect on theproduction of the pro-inflammatory cytokine TNF. TNF is released fromvarious cells (monocytes and B lymphocytes) following activation bymitogen or antigen. The production of this inflammatory mediator in thesupernatant of activated PBMCs was measured by ELISA.

Human PBMCs (2-5×10⁶ cells/well), purified from whole blood bycentrifugation through ficoll/hypaque, were incubated with either PWM(2.5 μg/ml) or PPD (1 μg/ml) and compound example 3 at 10 μM in a 24well microtitre plates for 48 hrs at 37° C. in a CO₂ incubator. DMSO wasrun in each assay as a vehicle control. Supernatant was removed after 48hrs and microfuged at 10,000 rpm for 2 minutes. Supematant was removed,aliquotted and stored at −70° C. until tested. The level of TNF wasmeasured by ELISA (R&D Sytems) and the results are shown in Table 2below.

TABLE 2 ACTIVATED ACTIVATED PWM PPD TNF (pg/ml) TNF (pg/ml) example 3495 15 dmso 1390 50

The compound of example 3 reduced the level of TNF production by 64% inthe PWM system and 70% in the PPD system.

In a modification of the above procedure, the compounds of Examples 19and 40 were tested at 10 μM for inhibition of TNF production inPWM-activated whole blood diluted 1/5 in RPMI containing 1% nutridoma.The supernatants were tested after 24 hours of culture. The results areshown in Table 3, from which it can be seen that both test compoundsreduced the level of TNF production.

TABLE 3 COMPOUNDS TNF alpha (10 μM) (pg/ml) EXAMPLE 19 489 EXAMPLE 40600 VEHICLE 1031

Biological Example E

The compound of Example 19 above was tested in an animal model ofautoimmune disease, known as Experimental Autoimmune Neuritis.

Male Lewis rats were inoculated with bovine myelin (10 mg.kg⁻¹) inFreunds adjuvant supplemented with Mycobacterium tuberculosis (7.5mg.kg⁻¹) subcutaneously at the base of the tail. Animals were randomizedby bodyweight into groups of 9. Dosing with the test compound (100mg.kg⁻¹ ip uid) or vehicle commenced on day 1 post-inoculation andcontinued through to day 14, the study ending on day 15. Animals wereweighed daily, and from day 9 onwards they were assessed using aclinical scoring system which reflects the severity of paralysis.

At day 15 post-inoculation there was a 93% reduction in mean clinicalscore of the drug treated group when compared to the vehicle group.

What is claimed is:
 1. A method for treatment of mammals suffering froma diseases responsive to inhibition of monocyte and/or macrophage and/orlymphocyte activation and of lymphocyte proliferation, comprisingadministering to the mammal suffering such disease an amount of acompound of general formula (I) or a pharmaceutically acceptable salthydrate or solvate thereof sufficient to inhibit such activation and/orproliferation:

wherein R is hydrogen or (C₁-C₆)alkyl; R₁ is hydrogen; (C₁-C₆)alkyl;(C₂-C₆)alkenyl; phenyl or substituted phenyl; phenyl (C₁-C₆)alkyl orsubstituted phenyl(C₁-C₆)alkyl; phenyl (C₂-C₆)alkenyl or substitutedphenyl(C₂-C₆)alkenyl heterocyclyl or substituted heterocyclyl;heterocyclyl(C₁-C₆)alkyl or substituted heterocyclyl(C₁-C₆)alkyl; agroup BSO_(n)A- wherein n is 0, 1 or 2 and B is hydrogen or a (C₁-C₆)alkyl, phenyl, substituted phenyl, heterocyclyl substitutedheterocyclyl, (C₁-C₆)acyl, phenacyl or substituted phenacyl group, and Arepresents (C₁-C₆)alkylene; hydroxy or (C₁-C₆)alkoxy; amino, protectedamino, acylamino, (C₁-C₆)alkylamino or di-(C₁-C₆)alkylamino; mercapto or(C₁-C₆)alkylthio; amino(C₁-C₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl,di(C₁-C₆)alkylamino(C₁-C₆)alkyl, hydroxy(C₁-C₆)alkyl,morcapto(C₁-C₆)alkyl or carboxy(C₁-C₆) alkyl wherein the amino-,hydroxy-, mercapto- or carboxyl-group are optionally protected or thecarboxyl-group amidated; lower alkyl substituted by carbamoyl,mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, di(loweralkyl)amino, or carboxy-lower alkanoylamino; or a cycloalkyl,cycloalkenyl or non-aromatic heterocyclic ring containing up to 3heteroatoms, any of which may be (i) substituted by one or moresubstituents selected from C₁-C₆ alkyl, C₂-C₆ alkenyl, halo, cyano(—CN), —CO₂H, —CO₂R, —CONH₂, —CONHR, —CON(R)₂, —OH, —OR, oxo-, —SH, —SR,—NHCOR, and —NHCO₂R wherein R is C₁-C₆ alkyl or benzyl and/or (ii) fusedto a cycloalkyl or heterocyclic ring; R₂ is a C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, phenyl(C₁-C₆ alkyl)-, heteroaryl(C₁-C₆ alkyl)-,phenyl(C₂-C₆ alkenyl)-, heteroaryl(C₂-C₆ alkenyl)-, phenyl(C₂-C₆alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, cycloalkyl(C₁-C₆ alkyl)-,cycloalkyl(C₂-C₆ alkenyl)-, cycloalkyl(C₂-C₆ alkynyl)-,cycloalkenyl(C₁-C₆ alkyl)-, cycloalkenyl(C₂-C₆ alkenyl)-,cycloalkenyl(C₂-C₆ alkynyl)-, phenyl(C₁-C₆ alkyl)O(C₁-C₆ alkyl)-, orheteroaryl(C₁-C₆ alkyl)O(C₁-C₆ alkyl)-group, any one of which may beoptionally substituted by C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, cyano (—CN),phenyl or heteroaryl, or phenyl or heteroaryl substituted by C₁-C₆alkyl, C₁-C₆ alkoxy, halo, or cyano (—CN); R₃ is the characterisinggroup of a natural or non-natural α amino acid in which any functionalgroups may be protected; and R₄ is an ester or thioester group.
 2. Theuse of a compound of formula (I) as defined in claim 1 in thepreparation of a composition for treatment of mammals suffering from adisease responsive to inhibition of monocyte and/or macrophage and/orlymphocyte activation and of lymphocyte proliferation.
 3. A method asclaimed in claim 1 wherein the stereochemical configuration of thecarbon atom carrying the group R₂ is R, and that of the carbon atomcarrying the groups R₃ and R₄ is S.
 4. A method as claimed in claim 1wherein R₁ is: hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl,hydroxyl, methoxy, allyl, phenylpropyl, phenylprop-2-enyl,thienylsulphanylmethyl, thienylsulphinylmethyl, orthienylsulphonylmethyl; or C₁-C₄ alkyl,eg methyl, ethyl n-propyl orn-butyl, substituted by a phthalimido,1,2-dimethyl-3,5-dioxo-1,2,4-triazolidin-4-yl,3-methyl-2,5-dioxo-1-imidazolidinyl,3,4,4-trimethyl-2,5-dioxo-1-imidazolidinyl,2-methyl-3,5-dioxo-1,2,4-oxadiazol-4-yl,3-methyl-2,4,5-trioxo-1-imidazolidinyl,2,5-dioxo-3-phenyl-1-imidazolidinyl, 2-oxo-1-pyrrolidinyl,2,5-dioxo-1-pyrrolidinyl or 2,6-dioxopiperidinyl,5,5-dimethyl-2,4-dioxo-3-oxazolidinyl,hexahydro-1,3-dioxopyrazolo[1,2,a][1,2,4]-triazol-2-yl, or anaphththalimido (ie 1,3-dihydro-1,3-dioxo-2H-benz[f]isoindol-2-yl),1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl,1,3-dihydro-1,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or2,3-dihydro-1,3-dioxo-1H-benz[d,e]isoquinolin-2-yl group; or cyclohexyl,cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl, cyclopropyl,tetrahydropyranyl or morpholinyl.
 5. A method as claimed in claim 1wherein R₁ is n-propyl, allyl, hydroxy, methoxy andthienylsulfanylmethyl.
 6. A method as claimed in claim 1 or the use asclaimed in claim 2 wherein R₂ is: C₁-C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆alkynyl; phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆alkynyl)- optionally substituted in the phenyl ring; heteroaryl(C₁-C₆alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆ alkynyl)-optionally substituted in the heteroaryl ring; 4-phenylphenyl(C₁-C₆alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-, 4-phenylphenyl(C₃-C₆ alkynyl)-,4-heteroarylphenyl(C₁-C₆ alkyl)-, 4-heteroarylphenyl(C₃-C₆ alkenyl)-,4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in theterminal phenyl or heteroaryl ring; or phenoxy(C₁-C₆ alkyl)- orheteroaryloxy(C₁-C₆ alkyl)- optionally substituted in the phenyl orheteroaryl ring.
 7. A method as claimed in claim 1 wherein R₂ is:methyl, ethyl, n- or iso-propyl, n-, iso- or tert-butyi, n-pentyl,n-hexyl, n-heptyl, n-nonyl, n-decyl, prop-2-yn-1-yl,3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, phenylpropyl,4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl,phenoxybutyl, 3-(4-pyridylphenyl)propyl-,3-(4-(4-pyridyl)phenyl)prop-2-yn-1-yl, 3-(4-phenylphenyl)propyl-,3-(4-phenyl)phenyl)prop-2-yn-1-yl, or 3-[(4-chlorophenyi)phenyl]propyl-.8. A method as claimed in claim 1 wherein R₂ is n- or iso-butyl,n-hexyl, cyclopentylmethyl, benzyl, and3-(2-chlorophenyl)prop-2-yn-1-yl.
 9. A method as claimed in claim 1wherein R₃ is C₁-C₆ alkyl, phenyl, 2-, 3-, or 4-hydroxyphenyl, 2-, 3-,or 4-methoxyphenyl, 2- or 3-thienyl, 2-, 3-, or 4-pyridylmethyl, benzyl,2-, 3-, or 4-hydroxybenzyl, 2-, 3-, or 4-benzyloxybenzyl, 2-, 3-, or4-C₁-C₆ alkoxybenzyl, or benzyloxy(C₁-C₆alkyl)-.
 10. A method as claimedin claim 1 wherein R₃ is the characterising group of a natural α aminoacid, in which any functional group may be protected, any amino groupmay be acylated and any carboxyl group present may be amidated.
 11. Amethod as claimed in claim 1 wherein R₃ is a group -[Alk]_(n)R₆ whereAlk is a (C₁-C₆)alkyl or (C₂-C₆)alkenyl group optionally interrupted byone or more —O—, or —S— atoms or —N(R₇)— groups [where R₇ is a hydrogenatom or a (C₁-C₆)alkyl group], n is 0 or 1, and R₆ is an optionallysubstituted cycloalkyl or cycloalkenyl group.
 12. A method as claimed inclaim 1 wherein R₃ is a benzyl group substituted in the phenyl ring by agroup of formula —OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy,phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino,phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid halide,ester or amide derivative thereof, said residue being linked via anamide bond, said amino acid being selected from glycine, α or β alanine,valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan,serine, threonine, cysteine, methionine, asparagine, glutamine, lysine,histidine, arginine, glutamic acid, and aspartic acid.
 13. A method asclaimed in claim 1 wherein R₃ is a heterocyclic(C₁-C₆)alkyl group,either being unsubstituted or mono- or di-substituted in theheterocyclic ring with halo, nitro, carboxy, (C₁-C₆)alkoxy, cyano,(C₁-C₆)alkanoyl, trifluoromethyl (C₁-C₆)alkyl, hydroxy, formyl, amino,(C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino, mercapto, (C₁-C₆)alkylthio,hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or (C₁-C₆)alkylphenylmethyl.14. A method as claimed in claim 1 wherein R₃ is a group—CR_(a)R_(b)R_(c) in which: each of R_(a), R_(b) and R_(c) isindependently hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl; or R_(c) is hydrogen and R_(a)and R_(b) are independently phenyl or heteroaryl such as pyridyl; orR_(c) is hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,phenyl(C₁-C₆)alkyl, or (C₃-C₆)cycloalkyl, and R_(a) and R_(b) togetherwith the carbon atom to which they are attached form a 3 to 8 memberedcycloalkyl or a 5- to 6-membered heterocyclic ring; or R_(a), R_(b) andR_(c) together with the carbon atom to which they are attached form atricyclic ring (for example adamantyl); or R_(a) and R_(b) are eachindependently (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,phenyl(C₁-C₆)alkyl, or a group as defined for R_(c) below other thanhydrogen, or R_(a) and R_(b) together with the carbon atom to which theyare attached form a cycloalkyl or heterocyclic ring, and R_(c) ishydrogen, —OH, —SH, halogen, —CN, —CO₂H, (C₁-C₄)perfluoroalkyl, —CH₂OH,—CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl,—SO(C₁-C₆)alkyl, —SO₂(C₁-C₆) alkyl, —S(C₂-C₆)alkenyl, —SO(C₂-C₆)alkenyl,—SO₂(C₂-C₆)alkenyl or a group —Q—W wherein Q represents a bond or —O—,—S—, —SO— or —SO₂— and W represents a phenyl, phenylalkyl,(C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl,(C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, whichgroup W may optionally be substituted by one or more substituentsindependently selected from, hydroxyl, halogen, —CN, —CO₂H,—CO₂(C₁-C₆)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl, —CONH(C₁-C₆alkyl)₂, —CHO,—CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl,—SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl,—N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl,(C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl orbenzyl.
 15. A method as claimed in claim 1 wherein R₃ is phenyl, benzyl,tert-butoxymethyl or iso-butyl.
 16. A method as claimed in claim 1wherein R₄ is a group of formula —(C═O)OR₉, —(C═O)SR₉, —(C═S)SR₉, and—(C═S)OR₉ wherein R₉ is is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, cycloalkyl,cycloalkyl(C₁-C₆)alkyl-, phenyl, heterocyclyl, phenyl(C₁-C₆)alkyl-,heterocyclyl(C₁-C₆)alkyl-, (C₁-C₆)alkoxy(C₁-C₆)alkyl- or(C₁-C₆)alkoxy(C₁-C₆)alkoxy(C₁-C₆)alkyl-, any of which may be substitutedon a ring or non-ring carbon atom or on a ring heteroatom, if present.17. A method as claimed in claim 1 wherein R₄ is a group of formula—(C═O)OR₉, wherein R₉ is methyl, ethyl, n- or iso-propyl, n-, sec- ortert-butyl, 1-ethyl-prop-1-yl, 1-methyl-prop-1-yl, 1-methyl-but-1-yl,cyclopentyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- and4-pyridylmethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1yl,adamantyl, tetrahydrofuran-3-yl or methoxyethyl.
 18. A method as claimedin claim 1 wherein R₄ is a group of formula —(C═O)OR₉ wherein R₉ isbenzyl, cyclopentyl, isopropyl or tert-butyl.
 19. A method as claimed inclaim 1 wherein R is hydrogen or methyl.
 20. A method as claimed inclaim 1 wherein R₁ is n-propyl, allyl, hydroxy, methoxy orthienylsulfanyl-methyl, R₂ is isobutyl, n-hexyl, cyclopentylmethyl,benzyl or 3-(2-chlorophenyl)prop-2-yn-1-yl, R₃ is phenyl, benzyl,tert-butoxymethyl, n-butyl or iso-butyl, R₄ is a group of formula—(C═O)OR₉ wherein R₉ is benzyl, cyclopentyl, isopropyl or tert-butyl andR is hydrogen or methyl.
 21. A method as claimed in claim 1 wherein thecompound is ary of those specified in any of the Examples 1 to 50herein.
 22. A method according to claim 1, wherein the disease to betreated is an autoimmune disease, transplant rejection,graft-versus-host disease, pancreatitis, or an allergy.